Methods of determining the sequence of nucleic acids are some of the most important tools in the field of molecular biology. Since the development of the first methods of DNA sequencing in the 1970s, sequencing methods have progressed to the point where a majority of the operations are now automated, thus making possible the large scale sequencing of whole genomes, including the human genome. There are two broad classes of DNA sequencing methodologies: (1) the chemical degradation or Maxam & Gilbert method and (2) the enzymatic or dideoxy chain termination method (also known as the Sanger method), of which the latter is the more commonly used and is suitable for automation.
Of particular interest in DNA sequencing are methods of automated sequencing, in which fluorescent labels are employed to label the size separated fragments or primer extension products of the enzymatic method. Currently, three different methods are used for automated DNA sequencing. In the first method, the DNA fragments are labeled with one fluorophore and then run in adjacent sequencing lanes, one lane for each base. See Ansorge et al., Nucleic Acids Res. (1987)15: 4593-4602. In the second methods, the DNA fragments are labeled with oligonucleotide primers tagged with four fluorophores and all of the fragments are run in one lane. See Smith et al., Nature (1986) 321: 674-679. In the third method, each of the different chain terminating dideoxynucleotides is labeled with a different fluorophore and all of the fragments are run in one lane. See Prober et al., Science (1987) 238: 336-341. The first method has the potential problems of lane-to-lane variations as well as a low throughput. The second and third methods require that the four dyes be well excited by one laser source, and that they have distinctly different emission spectra. Otherwise, multiple lasers have to be used, increasing the complexity and the cost of the detection instrument.
With the development of Energy Transfer primers which offer strong fluorescent signals upon excitation at a common wavelength, the second method produces robust sequencing data in currently commercial available sequencers. However, even with the use of Energy Transfer primers, the second method is not entirely satisfactory. In the second method, all of the false terminated or false stop fragments are detected resulting in high backgrounds. Furthermore, with the second method it is difficult to obtain accurate sequences for DNA templates with long repetitive sequences. See Robbins et al., Biotechniques (1996) 20: 862-868.
The third method has the advantage of only detecting DNA fragments incorporated with a terminator. Therefore, backgrounds caused by the detection of false stops are not detected. However, the fluorescence signals offered by the dye-labeled terminators are not very bright and it is still tedious to completely clear up the excess of dye-terminators even with AmpliTaq DNA Polymerase (FS enzyme). Furthermore, non-sequencing fragments are detected, which contributes to background signal. Applied Biosystems Model 373 A DNA Sequencing System User Bulletin, November 17,P3, August 1990.
Thus, there is a need for the development of improved methodology which is capable of providing for highly accurate sequencing data, even for long repetitive sequences. Such methodology would ideally include a means for isolating the DNA sequencing fragments from the remaining components of the sequencing reaction mixtures such as salts, enzymes, excess primers, template and the like, as well as false stopped sequencing fragments and non-sequencing fragments resulting from contaminated RNA and nicked DNA templates.
Relevant Literature
Methods of DNA sequencing are reviewed in Griffin and Griffin, Applied Biochemistry and Biotechnology (1993) 38: 147-159.
The effect of different labeling methodologies in automated DNA sequencing is discussed in Perkin Elmer User Bulletin (August 1990, Number 17) entitled "guide to Interpretation of 373A Dye Primer and Dye Terminator Data.
The use of biotinylated nucleotides in various sequencing applications is described in U.S. Pat. Nos. 5,484,701; 5,405,746 and 5,401,632, as well as in the following literature references: Yu et al., J. Biolumin. Chemilumin. (1995) 10: 239-245; Tong & Smith, J. DNA Sequencing and Mapping (1993) 4: 151-162; Wahlberg et al., Electrophoresis (1992) 13: 547-551; Tong & Smith, Anal. Chem. (1992) 64:2672-2677; Livak et al., Nuc. Acids. Res. (1992) 18: 4831-4837; Wahlberg et al., Molecular and Cellular Probes (1990) 4: 285-297; Wahlberg et al., Proc. Natl. Acad. Sci. USA (1990) 87: 6569-6573; Seliger et al., Nucleosides and Nucleotides (1990) 9:383-388; Beck, Methods of Enzymology (1990) 184: 612-617; Richterich, Nuc. Acids Res. (1989) 17:2181-2186; Beck et al., Nuc. Acids Res. (1989) 17: 5115-5123; Stahl et al., Nuc. Acids. Res. (1988) 16: 3025-3038.